Oxidized cardiolipin as a pro-inflammatory factor

ABSTRACT

Low levels of antibodies reactive with oxidised Cardiolipin (oxCL) in mammals are related to an increased risk of developing cardivacular diseases, auto-immune diseases or inflammatory conditions. High levels can have a protective function and in general there is a negative association between manifestations of these conditions and antibodies against oxCL. Thus, based on their relations methods of monitoring, determining and diagnosing as well as methods of immunisation and therapy of these diseases and conditions are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.13/1964,581 filed Aug. 12, 2013, which is a divisional of U.S.application Ser. No. 13/140,906, filed Jun. 20, 2011, which is a § 371National Stage of International Application No. PCT/EP20091009199 filedDec. 21, 2009, which claims benefit of U.S. Provisional Application No.61/138,966, filed Dec. 19, 2008. The entire text of each of theabove-referenced disclosures is specifically incorporated herein byreference without disclaimer.

FIELD OF THE INVENTION

The present invention relates to methods of monitoring, determining, orinfluencing the level of antibodies against oxidized cardiolipin(anti-oxCL) in a bodily fluid from a mammal. It is shown that low levelof anti-oxCL is correlated with a higher risk of developingcardiovascular disease, auto-immune diseases or inflammatory conditions.Thus, the invention relates also to a kit and the use of anti-oxCL as adiagnostic marker for determining the risk of developing acardiovascular disease, an auto-immune disease or an inflammatorycondition. Further, the invention relates to the use of agents foractivation immunotherapy and for the manufacture of a medicament fortreating, preventing, and/or reducing the risk of developing acardiovascular disease, an auto-immune disease or an inflammatorycondition.

BACKGROUND OF THE INVENTION

Auto-immune disease and inflammatory conditions are major healthproblems in the Western world and increasingly in developing countries.These diseases include Rheumatic conditions like Rheumatoid arthritisand Systemic lupus erythematosus (example of autoimmune diseases), thelatter often described as a prototypic autoimmune disease, and in textbooks more than 80 autoimmune rheumatic diseases are described, cf. e.g.Harrison's Principles of Internal Medicine, 17th Edition, which herebyis incorporated by reference. In addition, conditions like inflammatorybowel disease, asthma and diabetes type 1 are examples of auto-immuneconditions.

Interestingly and importantly, atherosclerosis and its consequencesstroke, myocardial infarction, acute coronary syndromes and heartfailure during recent years have been demonstrated to be inflammatoryconditions, with activated inflammatory and immune competent cellstypically present in the atherosclerotic plaques.

Further, also dementia including Alzheimer's disease, is characterizedby chronic inflammation, which has also been demonstrated forosteoarthritis.

Treatment modalities against inflammation in these diseases vary, but ingeneral have not been successful, with Rheumatoid arthrititis as apossible exception and there is a clear need of new types of treatment.Further, prediction by use of biomarkers is not optimal, and in manycases there is a great need of new types of treatment.

Cardiolipin (CL) is a dimeric phospholipid which is known to be presentin eucarytic cells, bacteria and Archaebacteria but its functional roleis only partly known, (Schlame M., 2008)¹. It is more prevalent in cellswith high metabolic activity, like heart and skeletal muscle, andespecially in mitochondrial membranes. The presence of CL inmitochondria and bacteria is interesting from an evolutionary point ofview since mitochondria are likely to have a bacterial origin, (Martin.W et al, 2001)². Also lipoproteins including low density lipoprotein(LDL) contain CL in contrast to what has previously has been thought,and two thirds of CL are present in low density lipoprotein (LDL),(Deguchi et al, 2000)³.

CL has a unique dimeric structure, highly enriched in linoleic acidgroups susceptible to oxidation (Schlame, M. et at, 2000, and Chicco, AJ et al, 2007)^(4,5). It has been suggested to play a role in generationof an electrochemical potential for substrate transport and ATPsynthesis both in bacteria and mitochondria, (Belikova, N A. et al, 2007and Bosova L V, el at, 2007)^(6,7). CL that has undergone oxidation(oxCL) promotes delocalization and release of cytochrome c, predisposingto its release from mitochondria and the activation of the cell deathprogrammes, (Chicco, A J. Et al, 2007, Gonzalzez, F. et al, 2007 andNakagawa, Y., 2004)^(5,8,9).

Antibodies against CL (aCL) cause both venous and arterial thrombosis,and are known to be of major importance in rheumatic diseases,especially lupus erythematosus/SLE by promoting cardiovascular diseaseand venous thrombosis, (Frostegard, J., 2005)¹⁰ and very high levels ofaCL are also linked to cardiovascular disease CVD in the generalpopulation, (Hamsten, A. et al, 1986)¹¹.

Annexin A5 is a member of the Annexin superfamily and hasanti-thrombotic properties due to interaction with phospholipids,especially phosphatidylserine, and thus the coagulation cascade. It hasrecently been demonstrated that aCL decrease binding of Annexin A5 toendothelial cells and it has been suggested that Annexin A5 could haveanti-atherothrombotic properties in general, (Cederholm, A. et al,2005)¹². Further, aCL cross reacts with oxidized low density lipoprotein(oxLDL), (Vaarala, O. et al, 1993)¹³. Since oxLDL is likely to be ofmajor importance in atherosclerosis and is present in large amounts inthe atherosclerotic lesions, (Hansson, G K., 2005)¹⁴, the associationwith aCL could have clinical implications.

SUMMARY OF THE INVENTION

Cardiolipin (CL) is a phospholipid with an unusual dimeric phospholipidsstructure, present in mitochondria and bacteria, but also inlipoproteins. The properties of oxidized CL (oxCL) and inhibitoryeffects of Annexin A5, an antithrombotic agent interfering withphospholipids, are determined.

It has been found that oxCL in contrast to CL has pro-inflammatoryproperties of relevance in atherogenesis and also rupture ofatherosclerotic plaques. These effects could be inhibited by Annexin A5.The implications of these findings are discussed.

The surprising characteristics of oxCL are confirmed by the fact thatlow concentrations of antibodies to mammal oxCl are an effectiveindicator of a cardiovascular disease, an auto-immune disease orinflammatory condition. It is within this present invention found thatantibodies to this particular antigen does not develop sufficiently inpatients before the clinical onset of cardiovascular disease, anauto-immune disease or inflammatory condition and presently it is apreferred hypothesis that an insufficiently developed natural immunityagainst oxCL represents an underlying mechanism for development of suchdiseases.

In connection with the present invention mammals means all known mammalsand in particular mice, rats, rabbits, dogs, cats, cattle, horses andhuman.

In connection with the present invention bodily fluid means any naturalbodily fluid or secretion of fluid including but not limited to plasma,serum, blood, urine, or saliva.

In connection with the present invention cardiovascular disease, anauto-immune disease or inflammatory condition means any of—including butnot limited to—the following diseases: cardiovascular disease (CVD),diabetes II, Alzheimer's disease, dementia in general, rheumaticdiseases, atherosclerosis, high blood pressure, acute and/or chronicinflammatory conditions, myocardial infarction, acute coronary syndrome,stroke, transient ischemic attack (TIA), claudiction, angina, type Idiabetes, rheumatoid arthritis, psoriasis, psoriatic arthritis,ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus,dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiplesclerosis, myasthenia gravis, asthma, encephalitis, inflammatory boweldisease, chronic obstructive pulmonart disease (COPD), arthritisincluding osteoarthritis, idiopathic inflammatory myopathies (IIM),dermatomyositis (DM), polymyositis (PM), inclusion body myositis, anallergic disorder and/or osteoarthritis in a mammal.

In connection with the present invention antibodies against oxCl(anti-oxCL) may be determined using any of the methods and techniquesconventional in the art for such determination. Conveniently, such amethod may comprise an immunoassay e.g. ELISA, EIA, immunofluorescence,Western blot, immunodiffusion, immunoelectrophoresis, immunoprecipitation and Magnetic immunoassay. Conveniently, the componentsneeded to perform the immunoassay will be supplied in a kit form.

In connection with the present invention a medicament may be intendedfor parenteral and mucosal administration and may be formulated withexcipients normally employed for such formulations. The medicament maybe administered in a way so as to be compatible with the dosageformulation and in such amount as will be therapeutically effective andimmunogenic.

In connection with the present invention vaccine means any agent that issuitable for increasing the anti-oxCL response, in particular oxCL orbioactive components and/or parts thereof optionally in combination withany suitable adjuvants.

In connection with the present invention monoclonal or polyclonalantibodies of isotype IgA, IgD, IgE, IgG, IgM, raised against oxCL orbioactive components and/or parts/fragments thereof refers to anymonoclonal or polyclonal antibody produced by immunisation of a suitablemammal, including but not limited to mouse, rabbit, goat, sheep, orhorse.

Also provided is method of treating, preventing or reducing the risk ofdeveloping a cardiovascular disease, an auto-immune disease orinflammatory condition in an mammal wherein said cardiovascular disease,an auto-immune disease or inflammatory condition is/are selected fromthe group comprising cardiovascular disease, diabetes II, Alzheimer'sdisease, dementia in general, rheumatic diseases, atherosclerosis, highblood pressure, acute and/or chronic inflammatory conditions, myocardialinfarction, acute coronary syndrome, stroke, TIA, claudiction, angina,type I diabetes, rheumatoid arthritis, psoriasis, psoriatic arthritis,ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus,dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiplesclerosis, myasthenia gravis, asthma, encephalitis, inflammatory boweldisease, chronic obstructive pulmonary disease (COPD), arthritis,idiopathic inflammatory myopathies (HM), dermatomyositis (DM),polymyositis (PM), inclusion body myositis, an allergic disorder and/orosteoarthritis, comprising administering to a mammal in need thereof atherapeutically effective amount of an agent that inhibits the activityof oxCL. The agent that inhibits the activity oxCL may be selected fromthe group consisting of Annexin A5, or a monoclonal or polyclonalantibody of isotype IgA, IgD, IgE, IgG, IgM, raised against oxCL orbioactive components and/or parts/fragments thereof, optionally incombination with any suitable adjuvants.

DETAILED DESCRIPTION OF THE INVENTION

The major underlying cause of CVD is atherosclerosis, which is nowadaysregarded as an inflammatory condition. However, it is mainly whenatherosclerotic plaques rupture or are otherwise damaged that CVDoccurs, typically at sites with an ongoing inflammatory process,(Hansson, G K, 2005 and Frostegard, J: et al 1999)^(14,16) . It istherefore of major importance to identify underlying proinflammatoryfactors in atherogenesis and CVD.

It has been found that oxidized CL, in contrast to native CL, haspro-inflammatory properties. The oxidation of native CL frok bovineheart was confirmed with mass spectrophotometer.

OxCL but not CL induced endothelial cells to express ICAM-1,intracelluiar adhesion molecule 1, and VCAM-1, vascular cell adhesionmolecule 1. Adhesion molecules play an important role to recruitmonocytes into interstitial in the artery, which is likely to be anearly step in atherogenesis and inflammatory process for development ofatherosclerosis.

Further, it is found and demonstrated that oxCL has the capacity toinduce interleukin 6, IL-6, production, but not CL. Several clinicalstudies have implicated three well-known markers of inflammation,C-reactive protein (CRP), fibrinogen (Fb) and IL-6, as CVD risk factors,and IL-6 has been identified as an independent risk factor for coronaryartery disease (CAD).

Furthermore, it is in this present invention reported that oxCL but notCL could inhibit Leucotrine B4, LTB4, production in both neutrophils andmacrophages. It is also found that OxCL but not CL can provokeinteracellular calcium mobilization which is an initiation signal forLTB4 production and also in general a sign of cell activation.Leukotrines are short-lived lipid mediators that have potentpro-inflammatory biological activities. Leukotriene B4 (LTB4) is one ofthe most potent chemotactic agent for other inflammatory cells and isbiosynthesized from arachidonic acid by the sequential action of5-lipoxygenase (5-LO) and Leucotrine A4, LTA4, hydrolase, mainly incells of myeloid lineage, such as neutrophil and macrophage, (Funk, CD., 2005)¹⁶. Two G-protein coupled LTB4 receptors have been identified,BLT1 and BLT2, with high and low affinity for LTB4, respectively,(Yokomizo, T. et al, 1997 and Yokomizo, T. et al, 2000)^(17,18). LTB4 isknown to exert broad pro-inflammatory effects, and evidence isaccumulating regarding the antimicrobial functions of LTB4, (Serezani, CH. Et al, 2005 and Wan, M. et al, 2007)^(19,20). Furthermore, theLTB4-BLT1 pathway was found to be important for linking early immuneresponses and the multiple classes of effector cells associated withacquired immunity, (Goodarzi, K. et al, 2003)²¹. LTB4 may play animportant role in atherosclerosis and CVD since mRNA levels for thethree key proteins are significantly increased in human atheroscleroticplaque, and more pronounced in patients with ongoing CVD, (Qui, H. etal, 2006)²².

Thus, by inducing LTB4, oxCL could therefore play a role in initiatingplaque rupture and CVD. Another finding herein is that oxCL but not CL(or reduced CL) inhibits uptake of oxLDL in macrophages.

OxLDL is taken up through specific scavenger receptors, which are notdown-regulated when exposed to increasing amounts of oxLDL (as opposedto the uptake of LDL), (Hansson, G K., 2005)¹⁴. Inhibition of thescavenger function is generally believed to be atheroprotective,preventing foam cell formation in the vascular wall which is a keyprocess in development of atherosclerosis. In line with this, micedefective in scavenger receptor function develop less atherosclerosis ascompared to control mice, (Febralo, M. et al, 2000)²³. It should benoted, however, that recent research indicates that different scavengerreceptors may play different roles and the role of scavenger receptorsmay vary depending on disease stage and type, (Moore, K J. et al,2006)²⁴.

If oxCL is predominantly exposed on oxLDL, binding and uptake of oxLDLcould through oxCL could promote atherogenesis. On the other hand, ifoxCL is present mainly on other compounds, e.g. apoptotic cells, otherproteins, or even bacteria, it is not clear how this would influencefoam cell development, which in principle could be decreased. However,since apoptotic cells are known not to have any pronouncedproinflammatory effects, this would suggest at least that oxCL is not animportant factor exposed during apoptosis. Further research is needed toclarify which parts of oxLDL play the largest role in foam cellformation.

It has recently been demonstrated that Annexin A5 is abundant inatherosclerotic lesions and that aCL can interfere with its binding toendothelial cells, promoting CVD in SLE, (Cederholm, A. et al, 2005)¹².Rand et al have demonstrated that Annexin A5 can form a crystallineshield over cell surfaces, which could have a protective function.However, this can be disrupted by aCL, causing the antiphospholipidantibody syndrome, characterized by arterial and venous thrombosis andalso miscarriage, (Rand, J H. Et al, 1999)²⁵. Annexin A5, ANXAS, hasrecently been implicated in CVD in general also as indicated by itsfunction as a potent antiatherothrombotic agent in a rabbit model ofarterial thrombosis, by interfering with tissue factor expression and byrecovery of hypercoagulability, (Cederholm, A. et al, 2005, Thiagarajan,P. et al, 1997 and Ishii, H. et al, 2007)²⁶⁻²⁸.

In connection with the present invention novel anti-inflammatoryproperties of Annexin A5, with potential interest in atherosclerosis andCVD are reported. Annexin A5 inhibited the proinflammatory effects ofoxCL, including induction of adhesion molecules, IL-6 The mechanismcould be that Annexin A5 binds to phosphatidylserine (PS) of endothelialcells, PS being a prothrombotic factor. It is also demonstrated thatAnnexin A5 can bind to OxCL but not CL, though the exact mechanisms arenot clear. Annexin A5 can thus in principle prevent these oxCL-inducedeffects by interacting with oxCL, though the exact mechanisms remain tobe shown. Compatible with this is the unusual properties of Annexin A5,enabling it to form crystalline layers, which by themselves could be ofanti-inflammatory, inhibiting pro-inflammatory effects of oxCL by amechanistic barrier.

CL is synthesized in cells de novo through the action of cardiolipinsynthases which is most active in high-metabolic tissue (where CL itselfis most abundant). In mitochondria, CL modification and remodellingoccurs including substantial changes in the acyl composition¹.Accumulating evidence now also suggests that remodeling defects of CLcould play a role in physiology and also pathology such as in diabetes,heart failure and Barth's syndrome, (Han, X. et al, 2007, Sparagna, G C.Et al, 2007 and Schlame, M, 2002)²⁹⁻³¹. In an interesting report it wasdemonstrated that CL is quickly oxidized when coated on ELISA-plates fordetermination of aCL and many aCL in fact recognize oxidized CL (oxCL) ,(Horkko, S. et al, 1996)³². oxCL being an antigen for aCL could thuscontribute to the antiphospholipid antibody syndrome, (Horkko, S. et al,1996)³². The nature of these antibodies is still debated, almost threedecades after their discovery, and it is likely that some aCL recognizeplasma protein co-factors binding to CL, but that others recognize thephospholipid moiety, (Frostegard, J., 2005)¹⁰.

Based on the findings herein, it is hypothesized that CL also couldcontribute to human chronic inflammatory disease in general and CVD inparticular, by its proinflammatory effects. CL binds easily to proteins,and it is possible that such complexes can become proinflammatory e.g.if exposed to the hypoxic and/or prooxidant environment inatherosclerotic plaques, but in principle also in other chronicinflammatory conditions like rheumatoid arthritis (RA) and SLE.

Interestingly, oxLDL and foam cells are present in rheumatoid arthritis,RA-synovia, (Winyard, P G. et al, 1993)³³, and oxLDL is raised andassociated with disease activity in RA (unpublished observation). Insystemic lupus erythematosus (SLE), the risk of CVD is very high due toa combination of traditional and non-traditional risk factors,(Frostegard, J., 2005)¹⁰. Both aCL and oxLDL are important examples ofnon-traditional risk factors. In addition to being raised in SLE per se,oxLDL is CVD in SLE, (Frostegard, J., 2005 and Frostegard, J. et al,2005)^(10,34). Since CL is easily oxidized, it could play a role inthese chronic inflammatory conditions.

It has recently been demonstrated that low levels of natural antibodiesagainst phosphorylcholine (anti-PC), another exposed antigen in oxLDL,are risk factors for development of CVD in men, (Sjöberg, B G, 2008)³⁵.phophorycholine, oxCL belongs to a novel class of pathogen-associatedmolecular patterns (PAMPs) and natural antibodies against oxCL bindoxLDL, (Tuominen, A, 200)³⁶. Thus, it is not known if there are commonpatterns of recognition in oxCL and PC, but the possibility that naturalantibodies against oxCL like anti-PC, are risk factors for CVD at lowlevels deserves further study.

Taken together, the findings of the present invention indicate that oxCLcan be a novel pro-inflammatory factor, causing or promoting plaquerupture in CVD and potentially also playing a role in other chronicinflammatory conditions including RA and SLE. Further, based on itscapacity to inhibit oxCL-effects, it can be hypothesized that Annexin A5also known as ANXA5, could be developed into a therapeutic agent inatherothrombosis and plaque rupture and also in other inflammatoryconditions where oxCL plays a role.

Immunoassays

Detection of antibodies is a common form of medical diagnostics. Forexample, in different biochemical assays for disease diagnosis, a titerof antibodies indicative for a particular disease is estimated from theblood and if those antibodies are not present, the person does not havethe disease in question.

Several immunodiagnostic methods based on detection of complexantigen-antibody are used to diagnose infectious diseases, for exampleELISA, immunofluorescence, Western blot, immunodiffusion,immunoelectrophoresis, and Magnetic immunoassay. Targeted monoclonalantibody therapy has been used to treat diseases such as rheumatoidarthritis, multiple sclerosis, psoriasis, and many forms of cancerincluding non-Hodgkin's lymphoma, colorectal cancer, head and neckcancer and breast cancer.

An immunoassay is a test that measures the concentration of a substancein a bodily fluid, using the reaction of an antibody or antibodies toits antigen. Both polyclonal and monoclonal antibodies can be used.Monoclonal antibodies usually bind only to one site of a particularmolecule, and therefore provide a more specific and accurate test. Boththe presence of antigen or antibodies can be measured.

In many applications, the response of the bodily fluid being measured iscompared to standards of a known concentration. This can be done throughthe plotting of a standard curve on a graph, the position of the curveat response of the unknown is then examined, and so the quantity of theunknown can be determined.

The most common method used for detecting the quantity of antibody orantigen is to label either the antigen or antibody. The label mayconsist of an enzyme (enzyme immunoassay (EIA, also called Enzyme-linkedimmunosorbant assay or ELISA), colloidal gold (lateral flow assays),radioisotopes such as I-125 Radioimmunoassay (RIA), magnetic labels(magnetic immunoassay—MIA) or fluorescence. Other techniques includeagglutination, nephelometry, turbidimetry and Western Blot.

Immunoassays are normally divided into those that involve labelledreagents and those which involve non-labelled reagents. Those whichinvolve labelled reagents can be subdivided into homogenous andheterogeneous immunoassays. Heterogeneous immunoassays can furthermorebe competitive or non-competitive. In simple terms, in ELISA or EIA anantigen is affixed to a surface, and then an antibody is washed over thesurface so that it can bind to the antigen. This antibody is then linkedto an enzyme (e.g. through binding with an additional antibody), and inthe final step a substance is added that the enzyme can convert to somedetectable signal.

ELISA

Thus, any form of ELISA (it being direct ELISA, indirect ELISA, sandwichELISA, competitive ELISA or reverse ELISA) can be performed to evaluateeither the presence of antigen or the presence of antibody in a sample,and is consequently a useful tool for determining serum antibodyconcentrations.

The ELISA, or the enzyme immunoassay (EIA), has a high sensitivity.Thus, in an ELISA of relevance for the present invention, a sample ofbodily fluid, e.g. a person's serum, can e.g. be diluted 400-fold andapplied to a plate to which oxCL is attached. If antibodies to oxCL arepresent in the serum, they may bind to oxCL. The plate can then bewashed to remove all other components of the serum. A specially prepared“secondary antibody”—an antibody that binds to the oxCL/anti-oxCLcomplex—can then be applied to the plate, followed by another wash. Ifthis secondary antibody is chemically linked in advance to an enzyme,the plate will contain enzyme in proportion to the amount of secondaryantibody bound to the plate. A substrate for the enzyme can then beapplied, and catalysis by the enzyme leads to a change in color orfluorescence. ELISA results can then be reported as a number, which cansubsequently be compared to the relevant “cut-off” point between apositive and negative result.

A cut-off point may be determined by comparing it with a known standard.This can be determined by applying a sample of known oxCL concentrationto a surface and fixing it to the surface to render it immobile. Samplesof known oxCL concentrations can then be used to generate a standardcurve.

Activation Immuno-Therapies

Immunotherapy is normally defined within medicine as “Treatment ofdisease by inducing, enhancing, or suppressing an immune response”.

Passive immunity can be achieved through the transfer of ready-madeantibodies into the affected individual. Immunotherapies designed toelicit or amplify an immune response are normally termed ActivationImmunotherapies.

Immunotherapies designed to reduce, suppress or more appropriatelydirect an existing immune response, as in cases of autoimmunity orallergy, are normally termed Suppression Immunotherapies. The activeagents of immunotherapy are collectively called immunomodulators.

Cut-Off Value

The average level of anti-oxCL in mammals depends on the type of bodilyfluid sample, the specific species, and may vary between the differentpopulation groups.

A cutoff value may be chosen so that concentrations of anti-oxCL lowerthan said cutoff value is associated with an increased risk ofdeveloping a cardiovascular disease, an auto-immune disease orinflammatory condition.

Thus, a suitable cut-off value for having a higher risk of developing acardiovascular disease, an autoimmune disease and/or an inflammatorycondition may be chosen to be 95%, 90%, 85%, 80%, 75%, 65%, 60%, 55%,50%, 45%, 40%, 35%, 30%, 25% or even less, of the average levels ofanti-oxCL usually present in the population, preferably less than 75%,more preferably less than 50% or even more preferably less than 33% or25% of the average levels of anti-oxCL found in the given population.

A cutoff value may be chosen so that concentrations of anti-oxCL higherthan said cutoff value is not associated with an increased risk ofdeveloping a cardiovascular disease, an auto-immune disease orinflammatory condition.

Thus, a suitable cut-off value for having a low risk of developing acardiovascular disease, an autoimmune disease and/or an inflammatorycondition may be chosen to be 30%, 40%, 50%, 60%, 70%, 76%, 78%, 80%,82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% of the average levels ofanti-oxCL usually present in the population, preferably more than 30%,more preferable more than 50%, even more preferably more than 76% of theaverage levels of anti-oxCL found in the given population.

Embodiments of the Invention

In one embodiment the invention relates to a method for monitoring ordetermining the level of oxCL-antibodies (anti-oxCL) in a bodily fluidfrom a mammal.

Thus, in a particular embodiment of the invention it relates to animmunoassay making use of oxCL or parts thereof and a marker ofantibody-origin for binding to anti-oxCL/oxCL-complexes comprising ameans for acquiring a quantity of the bodily fluid, a media havingaffixed thereto a capture antibody capable of complexing with anti-oxCL,and an assay for the detection of a complex of the capture antibody andthe anti-oxCL. The immunoassay may preferably be ELISA, most preferablysandwich ELISA.

In a further embodiment the invention relates to the use of anti-oxCL indiagnostic in vitro methods as a diagnostic marker of mammal diseaseswherein the level of anti-oxCL in a sample of bodily fluid from saidmammal is used in the diagnosis of the risk of said mammal developing acardiovascular disease, an auto-immune disease or inflammatorycondition.

In a further embodiment the invention relates to a kit for detecting thepresence of anti-oxCL in a bodily fluid said kit comprising a means foracquiring a quantity of the bodily fluid, a media having affixed theretoa capture antibody capable of complexing with anti-oxCL, and an assayfor the detection of a complex of the capture antibody and theanti-oxCL.

In a further embodiment the invention relates to the use of an agentthat inhibits the activity of oxCL for the manufacture of a medicamentfor treating, preventing and/or reducing the risk of developing acardiovascular disease, an auto-immune disease or inflammatory conditionin a mammal wherein the agent that inhibits the activity of oxCL isselected from the group consisting of Annexin A5, or a monoclonal orpolyclonal antibody of isotype IgA, IgD, IgE, IgG, IgM, raised againstoxCL or bioactive components and/or parts/fragments thereof.

In a further embodiment the invention relates to the use of oxCL orparts thereof for the manufacture of a medicament for use in activationimmunotherapy in the treatment, prevention and/or reduction of the riskof developing a cardiovascular disease, an auto-immune disease orinflammatory condition in a mammal.

In a further embodiment the invention relates to a method of treating,preventing or reducing the risk of developing a cardiovascular disease,an auto-immune disease or inflammatory condition in an mammal whereinthe agent that inhibits the activity of oxCL is selected from the groupconsisting of Annexin A5, or a monoclonal or polyclonal antibody ofisotype IgA, IgD, IgE, IgG, IgM, raised against oxCL or bioactivecomponents and/or parts/fragments thereof.

oxCL is a novel pro-inflammatory factor which plays a role in CVD and ininflammatory conditions and autoimmune diseases. Annexin A5 inhibits theeffects of oxCL and could be developed into a therapeutic agent inatherothrombosis and plaque rupture and also in other inflammatory andautoimmune conditions where oxCL plays a role.

Experimental

The materials and methods and examples disclosed below are provided onlyfor the purpose of illustrating the present invention and should not beconsidered as any limitation of the scope as outlined in the appendedclaims.

Materials and Methods

Chemical Treatments of Cardiolipin

Cardiolipin from bovine heart was purchased as ethanol solution fromSigma (Sigma product C 1649) and was stored at −20° C. Hydro heartcardiolipin (reduced CL) was purchased from Avanti Polar Lipids, Inc. Togenerate saturated molecular species, cardiolipin was oxidized inaqueous solutions containing 1.5 mmol/L tert-butylhydroperoxide andCuSO4 in concentrations ranging 20 umol/L. Both cardiolipin and copportreated cardiolipin were measured with MS-spectrophotometer, and it wasconfirmed to have been oxidized by copper and tert-butylhydroperoxide.

Endothelial Cells Culture and Adhesion Molecular

Pooled human umbilical vascular endothelial cells (HUVECs) at passage 2were purchased from Cascade Biologics, Inc (Portland, Oreg.). Cultureswere maintained in EGM™ phenol red-free medium (Clonetics, San Diego,Calif.), containing 2% of fetal bovine serum and supplements, at 37° C.under humidified 5% CO₂ conditions. All experiments were performed atpassage 3 to 5. HUVECs were seeded at 6×10⁴ cells/2mL density on 6-wellplates (NUNC Inc, Naperville, Ill.). After allowing endothelial cellsovernight for attachment, cells were ready for stimulation.

After washing with PBS, OxCL and reduced CL (20 ug/ml) were added. Forannexin A5 (From Bender MedSystems GmbH, Austria) inhibition study, OxCLwas incubated with Annexin A5 (10 ug/ml) for half hour before adding tocells. After 24 hours incubation, detached floating cells were washedaway, cell were harvested into Falcon FACS tubes. After centrifuging at1200 rpm for 5 minutes, cells were resuspended in 300 ul FACS buffer (1%FBS-PBS), incubated with 10p1 PE-conjugated anti-CD54 (eBioscience) and10 μl FITC-conjugated anti-Human CD106 (Becton, Dickinson) for 30minutes on ice. The intercellular adhesion molecule (ICAM-1) CD54 andthe vascular cell adhesion molecule (VCAM-1) CD106 were studied withflow cytometry analysis equipped with CellQuest software. For eachsample, 10,000 events were analyzed.

Macrophage Differentiation and Complement Induction

The macrophages which transformed from Human monocyte-derived THP-1cells (American Type Culture Collection, Manassas, Va., U.S.A.) wereplated in a 6-well plate at density of 1×106 cells/well in DMEM(INVITROGEN, USA) containing 10% FBS overnight. Then the cells werewashed three times with serum free medium before incubated with OxCL (2ug/ml), thereafter, the cells were washed ice-cold 4 times with 0.2%BSA/PBS and once with PBS. The cells were harvested in PBS containing0.1% BSA and 0.01% NaN3. 10 ul monoclonal mouse anti-human C5b-9 (FromDakocytomation Demark) were added after incubation 30 minutes on ice,cells were centrifugated at 4° C. and supernatant were discarded, PBScontaining 0.1% BSA and 0.01% NaN3 were added and 10 ul polyclonalrabbit anti-mouse IgG-FITC (Fab′2) (From Dakocytomation Demark) wereadded. Complement production was measured by flow cytometry (BDBiosciences, San Jose, Calif., USA). For each sample, minimum of 10.000events was analyzed.

IL-6 Production

HUVEC cells from above were seeded at density 10⁶/2ml into 6 wellplates. Allowing cells attachment for 24 hours, OxCL (20 ug/ml) with orwithout Annexin A5 (20 ug/ml) and reduced CL were added and incubatedfor 24 hours. Cell supernatant were collected and IL-6 and IL-8production were measured with protein multiplex immunoassay kits (fromBioscource, USA) and Bio-Plei™ system (BioRad, USA).

Enzyme-Linked Immunosorbent Assay (ELISA) for Annexin A5 Binding

F96 microtiter polysorp plates (Roskilde Denmark), were coated withOxCL, CL or Hydro Heart cardiolipin (Biosearch Technologies, Inc,Calif., USA) 10 λg/ml incubated overnight at 4° C. After five washingswith PBS, the plates were blocked with 2% PBS-BSA for 2h at roomtemperature. Annexin A5 were added and incubated for 1 hour. Afterwashing, bound annexin A5 was detected by incubating subsequently withrabbit anti-human annexin V polyclonal antibodies (Hyphen Biomed,Andresy, France) 1:2000 and polyclonal goat anti-rabbitImmunoglobulins/AP (DakoCytomation) 1:3000 were used. The reaction wasdeveloped with alkaline phosphatase substrate(Sigma), and opticaldensity (OD) was read at 405 nm with an ELISA Multiscan Plusspectrophotometer (Molecular Devices Emax, San Francisco). All sampleswere measured in duplicates and the coefficient of variation was below15%.

Use of other suitable monoclonal or polyclonal antibodies for detectingbound annexin V is also possible.

Labeling OxLDL with Dil

OxLDL (Industrylane Frederick) were incubated with Dil (Molecular ProbesEngene, Oregon, USA) in lipoprotein-deficient serum (Sigma) at 37° C.for 15 hours. Then dialyzed against saline-EDTA buffer for 6 hours.

Uptake of Dil-Labeled OxLDL

Uptake of Dil-labelled OxLDL was studied either by Flow cytometry orFluorescence/confocal microscopy. For microscopy, The macrophages 1×10⁶were grown overnight on culture slides (Nunc, Naperville, N.Y.), Forflow cytometry, The macrophages were plated in a 6-well plate at densityof 1×10⁶ cells/well in DMEM (INVITROGEN, USA) containing 10% FBSovernight. Then the cells were incubated with Dil-OxLDL (5 μg/ml), withOxCL (40, 80 μg/ml), with CL control (40, 80 μg/ml), with unlabeledOxLDL (40 μg/ml) or with unlabeled LDL (40 μg/ml) for 4 hours. ForAnnexin A5 inhibition of Dil-OxLDL experiment, macropages were incubatedwith Dil-Ox-LDL (5 ug/ml), with Annexin A5 (0.01, 0.04, 0.16, 0,64, 1,10, 20, 40 ug/ml). Thereafter, the cells from above were washed 4 timeswith 0.2% BSA/PBS and once with PBS. The cells were harvested in PBScontaining 0.1% BSA and 0.01% NaN3. Mean fluorescence intensity wasmeasured by flow cytometry (BD Biosciences, San Jose, Calif., USA). Foreach sample, fluorescence emission above 550 nm was measured and aminimum of 10.000 cells was analyzed.

Cell Culture

Human mononuclear cells were isolated from freshly prepared buffy coats(Karolinska Hospital blood bank, Stockholm, Sweden) by gradientcentrifugation on Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden).The mononuclear cells were cultured at a density of 5×10⁶/ml inRPMI-1640 medium with 25 mM Hepes, 1% L-glutamine, 1%penicillin-streptomycin and 10% FBS. After 7 days, there are proximate2×10⁶ macrophages per well.

Isolation of Polymorphonuclear Neutrophils (PMNs)

Human PMNs were isolated from freshly prepared buffy coats (KarolinskaHospital blood bank, Stockholm, Sweden) by dextran sedimentation,hypotonic lysis of erythrocytes and gradient centrifugation onLymphoprep (Axis-Shield PoC AS, Oslo, Norway). PMNs were suspended at adensity of 10×10⁶/ml in Dulbecco's PBS (Gibco (Invitrogen), Paisley,UK). PMN purity (>95%) and viability (>98%) was determined usingHemacolor (J. T. Baker, Utrecht, Holland) and Trypan Blue (SigmaChemical Co.) staining, respectively.

Intracellular Calcium Mobilization

Neutrophils were added into black, 96-well plates with transparentbottom (Corning Costar; 5×10⁴ cells/well), and spin down the plate at120×g for 3 min, afterwards changing the medium containing 4 μM FURA-2AM(Fura-2 acetoxymethyl ester), or buffer as appropriate, and the cellswere incubated for 30 min at 37° C. and 5% CO₂. Cells were washed fourtimes with 50 μl of a buffer solution (135 mM NaCl, 4.6 mM KCl, 1.2 mMMgCl₂, 1.5 mM CaCl₂, 11 mM glucose, 11 mM Hepes, pH 7.4) before a final45 μl volume of buffer was added to each well.

The plates were transferred to a fluorometer (Fluostar™, BMGTechnologies), and 50 μl of different agonists according to experimentaldesigns or buffer solution as control were injected into individualwells and the cells were monitored for the next 120 seconds. Controlwells containing cells that had not been exposed to FURA-2AM were usedto subtract background auto-fluorescence. The results are given as theratio of mean fluorescence intensity (MFI) between 340 and 380 nm, andnormalized by control.

Analysis of Leukotriene B₄ Biosynthesis by EIA Kit

2×10⁶ macrophages were incubated with different agents according to theexperimental design, and then quenched with an equal volume methanol.After acidified to pH 3-4, the samples were purified by solid-phaseextraction (Supelclean™ LC-18, Supelco) and eluted in methanol. Afterdried under nitrogen, the samples were re-suspended in EIA buffer. Thelevel of LTB₄ was determined with LTB₄ EIA kit (Cayman Chemical) byusing dilutions within the linear portion of the standard curve.

Ultrasound Scan for Assessing FMD and NTG Induced Vasodilatation

All ultrasound scans were made using a duplex scanner (Acuson Sequoia,Mountain View, Calif., USA) with a 8 MHz ART linear array transducer ina quiet semi-darkened room with the subject in the supine position.Scans were videotaped for off line analysis. Subjects were asked not tosmoke, and not to drink coffee or tea for at least 2 h before the study.All studies were done by the same operator using the same equipment. Allmeasures were done off-line from digitalised cine-loops by one measurer.

The brachial artery was scanned longitudinally 2-10 cm above the elbow,with the vessel placed horizontally across the screen. Settings weremade to optimise lumen-arterial wall interface, and thereafter notchanged during the study. The transducer was held in the same positionthroughout the study by a mechanical arm. A resting B-mode scan wasrecorded, flow velocity was measured with a 5 MHz pulsed Doppler with a1.5 mm gate width in the centre of the vessel at a 70 degree angle.Flow-increase was induced by the post ischemic response to deflation ofa pneumatic tourniquet placed around the forearm of the patient to 250mmHg for 4.5 min. A second scan was recorded from 30 s before to 90 safter cuff deflation. Flow velocity was recorded with a pulsed waveDoppler for 15 s before and 15 s after cuff deflation. This was followedby 10 minutes rest for vessel recovery. A third scan at rest was takenand 0.40 mg sublingual nitro-glycerine spray was administered. Three tofour minutes after nitro-glycerine the last scan was recorded. Thevessel was measured at a fixed point in all scans. Measures were made ondigitalised cone-loops from the anterior wall leading edge of theintima-media echo to the leading edge of the far wall intima-mediavessel interface incidentally with the R-wave on the ECG for threeconsecutive cardiac cycles and the average measurements were used. Afterreactive hyperaemia, measurements were made 50-70 s after cuffdeflation. The increase in vessel diameter during hyperemia and afternitro-glycerine administration is expressed as percentages of the firstcontrol scan.

Determination of Anti-OxCL Antibody Levels

The Immulon 1B plate was coated with 50 μl/well of OxCL 10 μg/ml, andallowed to dry overnight at 4° C. After washing with PBS, the plate wasblocked with 2% BSA at room temperature for 2 hours. 1:50 diluted serawere added in duplicates. The plate was incubated overnight at 4° C. Thesecondary antibody (anti-Ig) was added 100 μl/well, then left overnightat 4° C. Then five times Washing with PBS, Substrate was added 100μl/well, The ELISA Multiscan Plus Spectrophotometer was used todetermine optical density.

TNF Production

The PBMC were cultured in 96-well culture plates at density of2*10⁵cells/100 ul/well with stimulation of 1 ug/ml PHA in mediumpresence with different sera for 48 hours, and the supernatants weremeasured for TNF-a concentration by commercially available ELISA kits.

Extraction of Anti-OxCL IgG from Intravenous Immunoglobulin (IVIG)

OxCL-MBSA and MBSA were coupled to a HiTrap NHS column (AmershamBiosciences) separately according to the manufacturer instruction.

Human pooled immunoglobulin (IVIG; Gammaguard, S/D) was diluted inbinding buffer (20 mM Na₂HPO₄) at 50 mg/ml and filtered through 0.45 μmfilter before passing through pre-coupled OxCL-MBSA and MBSA Sepharosegel column. anti-OxCL IgG was eluted by 0.1M Glycin-HCl buffer. Thepurified fractions were desalted using PD-10 columns (Amersham PharmaciaBiotech AB). Binding to oxCL (as described for determination ofanti-oxCL antibodies) was confirmed.

Adhesion Molecule Expression by Endothelial Cells—Inhibition byExtracted Anti-OxCL

Pooled human umbilical vascular endothelial cells (HUVECs) at passage 2were purchased from Cascade Biologics, Inc (Portland, Oreg.). Cultureswere maintained in EGM™ phenol red-free medium (Clonetics, San Diego,Calif.), containing 2% of fetal bovine serum and supplements, at 37° C.under humidified 5% CO2 conditions. All experiments were performed atpassage 3 to 5. HUVECs were seeded at 6×104 cells/2mL density on 6-wellplates (NUNC Inc, Naperville, Ill.).

After allowing 24 hours for cells attachment, the cells were incubatedwith oxidized cardiolipin (oxCL) 10 μg/ml either with or withoutanti-oxCL-IgG 0.22 mg/ml. After 24 hours incubation, detached floatingcells were washed away, cell were harvested into Facoln FACS tubes.After centrifuging at 1400 rpm for 5 minutes, cells were resuspended in300 ul FACS buffer (1% FBS-PBS), incubated with 10 μl FITC-conjugatedanti-Human CD106 (Becton, Dickinson) for 30 minutes on ice. The vascularcell adhesion molecule (VCAM-1) CD106 were studied with flow cytometryanalysis equipped with CellQuest software. For each sample, 10,000 cellswere analyzed.

DRAWINGS

FIG. 1A and 1B: Electrospray ionization mass MS spectrometer (Micromass,Beverly, Mass.) was used to demonstrate that bovine heart cardiolipinwas oxidized.

FIG. 2A and 2B: Induction of adhesion molecules. Oxidized CL but not CLinduces adhesion molecules in endothelial cells, flow cytometry.

Endothelial cells were incubated with Oxidized CL (20 ug/ml) or CL (20ug/ml) for 24 hours, oxidized CL induced ICAM-1 and VCAM-1 productionbut CL does not show same effect.

FIG. 3A and 3B: inhibition of oxCL-induced adhesion molecule expressionby Annexin A5, detected by Flow cytometry.

Oxidized CL (20 ug/ml) was preincubated with Annexin A5 (10 ug/ml)before stimulating cells for 24 hours. Annexin A5 inhibited oxCL inducedendothelial cell expression of ICAM-1 (CD54) and VCAM-1 (CD106).

FIG. 4: Induction of IL-6 by oxCL and inhibition by Annexin A5.determined by Luminex. Detected by intensity of fluorescence.

Endothelial cells were incubated with OxCL (20 ug/ml) with or withoutAnnexin A5 (20 ug/ml) and CL (20 ug/ml) for 24 hours, oxidized CL cansignificantly induce IL-6 production and this effect can be inhibited byAnnexin A5. CL had no such effect.

FIG. 5: Binding of oxCL by Annexin A5, ELISA.

OxCL, CL and reduced CL (5 ug/ml) were coated on ELISA plates overnight.Annexin A5 (0.32 ug/ml, 0.64 ug/ml, 1.28 ug/ml, 2.56 ug/ml, 5.12 ug/mland 10.24 ug/ml) can bind to oxidized cardiolipin and air exposed CL butnot reduced cardiolipin.

FIG. 6A and 6B: Effect of oxCL on uptake of oxLDL in macrophages,detected by Flow cytometry.

OxCL competed macrophage uptake of OxLDL, but CL did not have sucheffect. (Human acute monocytic leukemia cell line) THP-1, cellsdifferentiated MQ were studied uptake of dil-OxLDL, the uptake could becompeted by OxCL but not non-oxidized CL.

FIG. 7A, 7B, 7C and 7D: oxCL-induced calcium mobilization, Fluorometer.

Results shows that only oxidized cardiolipin can activate theneutrophils and induce the intracellular calcium mobilization.Furthermore, annexin can prevent the effects of oxCL on neutrophils.

FIG. 8: Effect of oxCL on leukotriene B4 release, EIA.

Oxidized cardiolipin promotes human neutrophils and macrophages torelease leukotriene B₄, but cardiolipin did not show the similarreaction.

FIG. 9: Effect of Annexin A5 on oxCL induced LTB₄ production fromoxCL-stimulated human neutrophils and macrophages, which may give newinsights into clinical novel targets for medical treatments of theassociated inflammatory conditions, EIA.

FIG. 10: Antibodies against oxCL were extracted from human Ig andincubated with oxCL. Experiment indicates that such extracted anti-OxCLdecrease effects of oxCL, as seen by a shift to the left of thehistogram.

FIG. 11A, 11B, 11C, 11D, 11E and 11F: Effects of oxCL on activation ofT-cells, flow cytometry.

FIG. 11A: Effects on CD4-positive T-cells, control

FIG. 11B: Effects on CD4-positive T-cells, 5 μg/ml of oxCL

FIG. 11C: Effects on CD4-positive T-cells, 5 μg/ml of CL (native,non-oxidized CL)

FIG. 11D: Effects on CD8-positive T-cells, control

FIG. 11E: Effects on CD8-positive T-cells, 5 μg/ml of oxCL

FIG. 11F: Effects on CD8-positive T-cells, 5 μg/ml of CL

Human PBMC (Peripheral blood mononuclear leukocytes) were incubated overnight with 5 μg/ml of oxCL or CL. Both CD4 and CD8 positive T cells werestudied. Quadrant Q2 represent (%) T-cells positive for CD69 expressingand thus activated T-cells. Thus, it is demonstrated that oxCL but notCL can activate CD8-positive and CD4-positive T cells as determined byan increase in Q2 which is highly significant.

FIG. 12A, 12B and 12C: A Neuroblastoma cell line was treated withamyloid peptide 1-42 which induced cell death (as determined bypropidium iodide; PI). Extracted anti-oxCL inhibited the effect.

EXAMPLE 1 Chemical Treatments of Cardiolipin

Native cardiolipin and oxidation product were further analyzed by massspectrometry, FIG. 1A and 1B. Native cardiolipin from bovine heartyielded two major signals, corresponding to the double charged anion(m/z 724.0), the single charged anion (m/z 1447). The oxidizedderivative peaks from both double charged and single charged ions in theoxidized fraction were 8 m/z units apart, suggesting progressivelyoxidized cardiolipins.

EXAMPLE2 Endothelial Cells and Adhesion Molecules

To study whether oxCL can stimulate endothelial cells to expressadhesion molecules, HUVECs from passage 3 to 5 were incubated for 24 hwith OxCL or native CL. Results suggested oxCL can significantlyincrease both ICAM-1 and VCAM-1 expression. But native CL did not showthe same effect (FIG. 2A and 2B). Both the increased expression ofICAM-1 and VCAM-1 (CD54 and CD106) induced by oxCL were significantlyinhibited by Annexin A5 (FIG. 3A and 3B).

EXAMPLE3 IL-6 Production

10×6 HUVECs were seeded at 6 well plates. oxCL could stimulateendothelial cells produce 564.3±142.02 (pg/ml) IL-6 from the supernant.Annexin A5 significantly decreased H-6 level to 276.4±28.62 (pg/ml).Comparing with the control group, both native CL and Annexin A5themselves could not significantly increase endothelial cell II-6 levels(FIG. 4). This supports the hypothesis that oxCL stimulates the IL-6,which is a known inflammatory marker, e.g. oxCL might be involved ininducing/mediating inflammatory responses.

EXAMPLE4 Uptake Experiment

To study potential mechanisms of oxCL bioactivity, 1×10⁶/well THP-1differentiated macrophages (MQ) were used for uptake studies. oxLDLcould inhibit dil-OxLDL uptake up to more than 60%, but LDL hade almostno effect on the uptake of dil-oxLDL which confirmed the specificity ofuptake results. Further results suggested oxCL could in a concentrationdependent manner inhibit dil-oxLDL uptake while the native CL did notshow the competition effetcts (FIG. 6A and 6B).

EXAMPLE5 Intracellular Calcium Mobilization

To study the influence of oxCL on intracellular calcium mobilization,5×104 neutrophil cells/well were used for measuring calciummobilization. After adding oxCL, MFI of calcium mobilization in thecells were significantly increased within 120 seconds. This increaseinduced by oxCL was greatly inhibited by incubation of Annexin A5. ButAnnexin A5 and native CL did not increase calcium mobilization

(FIG. 7A-7D).

EXAMPLE6 LTB4 Production

Human mononuclear cells isolated from freshly prepared buffy coats weredifferentiated into macrophages according to protocols which are wellknown to those skilled in the art. 2×10⁶ cells/well were exposed toOxCL, CL or buffer solution. Results suggested OxCL could in aconcentration dependent manner induce LTB4 production but not withnative CL (FIG. 8). This increase was greatly inhibited by Annexin A5(FIG. 9).

EXAMPLE7 ELISA for Annexin A5 Binding to Antigen

To test potential protective mechanisms of Annexin A5, oxCL, CL andreduced CL (5 ug/ml) were coated on ELISA plates overnight beforedifferent concentrations of Annexin A5 (0.32 ug/ml, 0.64 ug/ml, 1.28ug/ml, 2.56 ug/ml, 5.12 ug/ml and 10.24 ug/ml) were added to plates.Results suggested that different concentration of Annexin A5 could bindto oxCL, which was also much stronger than overnights air exposed CL.Reduced CL could not bind to Annexin A5 (FIG. 5).

EXAMPLE8 The Role of IgM Antibodies Against Oxidized Cardiolipin(Anti-oxCL) in Prediction of Cardiovascular Disease (CVD) by Use ofELISA.

Between Jul. 1, 1997 and Jun. 30, 1998, every third 60-year old man andwoman, in the County of Stockholm, were invited to participate. Theresponse rate was 78% and 4232 subjects (2039 men and 2193 women) wereincluded. Information on sociodemography, lifestyle, medication anddiseases was obtained by a self-administered questionnaire.

Physical examination (including blood pressure measurements,anthropometry and ECG) was performed and serum, plasma and whole bloodwere collected for storage (−80° C.). Routine tests were used forfasting levels of blood lipids (total cholesterol, HDL-cholesterol,LDL-cholesterol and triglycerides), apolipoproteins blood glucose,s-insulin, p-fibrinogen and hsCRP.

New events of coronary heart disease (CHD), myocardial infarction (MI),hospitalization for angina pectoris, ischemic stroke were registered.The study base of 4232 subjects was matched with that of the nationalcause of death registry (fatal events until Dec. 31, 2003) and thenational in-hospital registry (non-fatal events until Dec. 31, 2005).

211 incident cases of CVD were recorded. Only subjects without a historyof CVD prior to recruitment were utilized for the matching procedures.To assess diagnoses of CVD events, the International Classification ofDiseases (ICD-10) was used to register CHD-deaths (I 20, I 21, I 46), MI(I 21), angina pectoris including PCIs and CABGs (I 20, Z 95.5 and Z95.1) and ischemic stroke (I 63-I 66). For each case three sex- andage-matched controls were randomly selected. Through this nestedcase-control design (211 cases and 633 controls) was applied todetermine relative risks for future events.

The screening of 4232 subjects, 60-year-old (2039 men and 2193 women)with a follow up of 5-7 years was conducted and 211 incident cases ofCVD (myocardial infarction, ischemic stroke, or hospitalized anginapectoris) and 633 age- and sex-matched controls were identified. Valueswere adjusted for smoking, BMI, type II diabetes, hypercholesterolaemia,and high blood pressure. An increased risk of CVD was determined asdescribed in the tables.

Crude values include the matching factors age and sex. In additionadjustments have been made for smoking, BMI>29 kg/m², type II diabetes,high s-cholesterol ≥5mmol/l, high blood pressure >140/90 mmHg.

Descriptive analyses of were performed for cases and controlsrespectively with values expressed as medians (ranges) or proportions.Relative risks (RR) with 95% confidence intervals (CI) were calculatedapplying conditional logistic regressions. Analyses were run both crudeand adjusted for traditional risk factors (as assessed by univariateanalyses of significant differences between cases and controls,).Statistical analyses were run with SAS®statistical software systemversion 9.1.

Thus, low anti-oxCL levels predicted a striking risk of stroke (CVD) andmyocardial infarction (MI) in men, and also increased risk of myocardialinfarction (MI) in women. It was not possible to study a similar effectof low anti-oxCL on the risk of stroke in woman due to a limited set ofdata.

Units were arbitrarily determined with sera two persons with moderatelyhigh-level anti-oxCL on each ELISA plate and these were used as controlsand was the basis of Unit determinations. Quartile 4 represents thehighest 25% of values in the whole cohort.

TABLE 1a Association between low levels of anti-OxCL and risk for CVD,men and women aOxCL Crude Adjust Quartiles RR 95% CI P-values RR 95% CIP-values Quartile 4 1 N/A N/A 1 N/A N/A Quartile 3 1.66 0.97-2.85 0.06531.84 1.08-3.33 0.0267 Quartile 2 1.67 0.97-2.88 0.0658 1.69 0.96-3.000.0699 Quartile 1 2.15 1.25-3.70 0.0036 2.15 1.23-3.78 0.0075

TABLE 3a Association between low levels of anti-OxCL and risk for MI,men aOxCL Crude Adjust Quartiles RR 95% CI P-values RR 95% CI P-valuesQuartile 4 1 N/A N/A 1 N/A N/A Quartile 3 1.39 0.73-2.66 1.6 0.81-3.17Quartile 2 1.51 0.78-2.94 1.7 0.84-3.46 Quartile 1 2.16 1.14-4.09 2.301.17-4.51

TABLE 4 Association between low levels of anti-OxCL and risk for MI,women aOxCL Crude Adjust Quartiles RR 95% CI P-values RR 95% CI P-valuesQuartile 4 1 N/A N/A 1 N/A N/A Quartile 3 2.52 0.93-6.82 2.96 1.05-8.30Quartile 2 2.13 0.82-5.52 1.97 0.73-5.31 Quartile 1 1.97 0.70-5.61 1.870.65-5.41

TABLE 5a Association between low levels of anti-OxCL and risk forstroke, men Crude Adjust aOxCL P- P- Quartiles RR 95% CI values RR 95%CI values Quartile 4 1 N/A N/A 1 N/A N/A Quartile 3 2.24 0.54-9.31  7.051.05-47.4 Quartile 2 2.7 0.54-13.48 8.03  0.93-69.56 Quartile 1 2.690.64-11.31 12.28  1.48-101.77

Relative risks (RR) using SAS were determined with 95% confidenceintervals (CI) and quartiles of anti-oxCL levels where the 100%percentile was set as the reference value (RR=1.0). Thus, the percentileunit represent the indicated fraktile of the level of anti-oxCL of theanalysed patients, respectively. Cut off levels are based on the totalof men and women.

Thus, low anti-oxCL levels predicted a striking risk of stroke (CVD) andmyocardial infarction, MI, in men, and also increased risk of myocardialinfarction, MI, in women. It was not possible to have a similar effectof low anti-oxCL on the risk of stroke in woman confirmed due to alimited set of data].

In the following tables in this example, levels below the indicated werecompared to those above.

TABLE 7a Association between low levels of anti-OxCL and risk for cvdmen + women aOxCL Crude Adjust percentile P- P- unit RR 95% CI values RR95% CI values 35 1.499 1.077 2.086 0.0163 1.520 1.076 2.146 0.0174 331.487 1.067 2.073 0.0191 1.484 1.049 2.099 0.0256 30 1.408 1.006 1.9710.0460 1.386 0.976 1.968 0.0684 25 1.361 0.951 1.949 0.0919 1.366 0.9411.982 0.1010 24 1.206 0.838 1.735 0.3136 1.219 0.836 1.776 0.3034 231.168 0.808 1.686 0.4086 1.185 0.810 1.733 0.3826 22 1.129 0.779 1.6360.5220 1.145 0.781 1.677 0.4882 21 1.204 0.831 1.745 0.3253 1.219 0.8311.787 0.3111 20 1.280 0.876 1.870 0.2018 1.282 0.867 1.895 0.2137 191.345 0.915 1.978 0.1315 1.333 0.896 1.983 0.1559 18 1.493 1.012 2.2021.480 0.991 2.211 0.0556 17 1.400 0.938 2.090 0.0993 1.379 0.913 2.0820.1265 16 1.385 0.915 2.097 0.1235 1.363 0.891 2.085 0.1531 15 1.4100.924 2.151 0.1108 1.386 0.901 2.132 0.1377 14 1.527 0.988 2.360 0.05681.511 0.969 2.357 0.0688 13 1.620 1.040 2.524 0.0330 1.556 0.989 2.4460.0558

TABLE 8a Association between low levels of anti-OxCL and risk for cvdmen aOxCL Crude Adjust percentile P- P- unit RR 95% CI values RR 95% CIvalues 35 1.936 1.295 2.896 0.0013 2.050 1.342 3.133 0.0009 33 2.0271.355 3.033 0.0006 2.142 1.401 3.274 0.0004 30 1.801 1.201 2.700 0.00441.885 1.232 2.884 0.0035 25 1.594 1.047 2.427 0.0296 1.655 1.067 2.5660.0244 24 1.401 0.913 2.148 0.1224 1.440 0.924 2.246 0.1073 23 1.3570.882 2.087 0.1648 1.412 0.903 2.208 0.1305 22 1.265 0.819 1.954 0.28951.311 0.837 2.053 0.2369 21 1.347 0.873 2.078 0.1777 1.389 0.887 2.1740.1511 20 1.412 0.910 2.189 0.1235 1.460 0.927 2.299 0.1026 19 1.4480.927 2.262 0.1037 1.479 0.932 2.346 0.0966 18 1.618 1.031 2.539 0.03621.638 1.026 2.613 0.0386 17 1.519 0.958 2.409 0.0754 1.507 0.937 2.4240.0909 16 1.570 0.973 2.533 0.0646 1.560 0.955 2.549 0.0760 15 1.5220.935 2.476 0.0910 1.485 0.903 2.442 0.1192 14 1.620 0.980 2.677 0.05981.591 0.952 2.660 0.0763 13 1.839 1.104 3.062 0.0193 1.731 1.028 2.9150.0392

TABLE 9a Association between low levels of anti-OxCL and risk for MI menaOxCL Crude Adjust percentile P- P- unit RR 95% CI values RR 95% CIvalues 35 1.895 1.213 2.960 0.0050 1.912 1.191 3.069 0.0073 33 2.0051.283 3.133 0.0023 2.023 1.259 3.250 0.0036 30 1.852 1.178 2.909 0.00751.829 1.135 2.947 0.0132 25 1.652 1.032 2.643 0.0363 1.609 0.980 2.6420.0602 24 1.508 0.936 2.429 0.0912 1.460 0.884 2.413 0.1396 23 1.4310.884 2.318 0.1449 1.409 0.846 2.347 0.1882 22 1.290 0.792 2.103 0.361.274 0.764 2.124 0.3525 21 1.397 0.859 2.270 0.1777 1.361 0.816 2.2680.2376 20 1.484 0.906 2.431 0.1169 1.442 0.859 2.422 0.1661 19 1.4770.895 2.438 0.1270 1.426 0.844 2.409 0.1851 18 1.614 0.978 2.663 0.06101.545 0.911 2.620 0.1062 17 1.561 0.937 2.599 0.0873 1.470 0.862 2.5090.1575 16 1.589 0.935 2.701 0.0871 1.511 0.870 2.622 0.1426 15 1.5290.889 2.630 0.1249 1.433 0.817 2.513 0.2097 14 1.658 0.954 2.882 0.07291.574 0.887 2.794 0.1212 13 1.895 1.080 3.327 0.0260 1.724 0.959 3.0980.0685

TABLE 10a Association between low levels of anti-OxCL and risk forstroke men aOxCL per- Crude Adjust centile P- P- unit RR 95% CI valuesRR 95% CI values 35 2.127 0.833 5.430 0.1143 4.031 1.107 14.674 0.034533 2.127 0.833 5.430 0.1143 4.031 1.107 14.674 0.0345 30 1.607 0.6454.003 3.410 0.942 12.347 0.0617 25 1.385 0.543 3.531 0.4950 2.453 0.7198.376 0.1521 24 1.042 0.397 2.735 0.9342 1.712 0.497 5.898 0.3941 231.104 0.426 2.866 0.8382 1.762 0.521 5.962 0.3622 22 1.174 0.452 3.0510.7421 1.882 0.553 6.406 0.3118 21 1.174 0.452 3.051 0.7421 1.882 0.5536.406 0.3118 20 1.174 0.452 3.051 0.7421 1.882 0.553 6.406 0.3118 191.344 0.506 3.565 0.5531 2.123 0.611 7.372 0.2358 18 1.637 0.584 4.5880.3488 2.246 0.638 7.907 0.2078 17 1.352 0.465 3.933 0.5802 1.704 0.4746.125 0.4145 16 1.491 0.495 4.490 0.4772 1.86 0.506 7.095 0.3423 151.491 0.495 4.490 0.4772 1.896 0.506 7.095 0.3423 14 1.454 0.441 4.7920.5388 1.566 0.388 6.321 0.5290 13 1.605 0.486 5.306 0.4379 1.649 0.4276.371 0.4679

EXAMPLE9

The same data material as described in example 8 was used herein.

Descriptive analyses were performed for cases and controls respectivelywith values expressed as medians (ranges) or proportions. Relative risks(RR) with 95% confidence intervals (CI) were calculated applyingconditional logistic regressions. Analyses were run both crude andadjusted for traditional risk factors (as assessed by univariateanalyses of significant differences between cases and controls,).Statistical analyses were run with SAS®statistical software systemversion 9.1.

Relative risks (RR) using SAS were determined with 95% confidenceintervals (CI) and quartiles of anti-oxCL levels where the highestquartile representing the highest values of anti-OxCL was set as thereference value (RR=1.0). Thus, percentile units represent the 76% andup to 98% fraktile of the level of anti-oxCL of the analysed patients,respectively. Cut off levels are based on the total of men and women.

Thus, low levels of anti-OxCL are associated with increased risk ofdevelopment of CVD. Here it is demonstrated that high levels ofanti-OxCL can be protective for CVD.

Association Between High Levels of Anti-OxCL and Risk for CVD(MI-Stroke) Men+Women

aOxCL Crude Adjust percentile P- P- unit RR 95% CI values RR 95% CIvalues 98 0.693 0.197 2.430 0.5662 0.566 0.154 2.078 0.3912 96 0.8250.354 1.924 0.6564 0.716 0.297 1.722 0.4550 94 0.649 0.310 1.360 0.25220.606 0.283 1.296 0.1966 90 0.631 0.351 1.134 0.1239 0.600 0.331 1.0890.0929 86 0.537 0.319 0.905 0.0196 0.485 0.283 0.829 0.0082 84 0.5760.356 0.932 0.0246 0.534 0.326 0.874 0.0125 82 0.607 0.387 0.950 0.02910.577 0.365 0.913 0.0187 80 0.698 0.460 1.057 0.0892 0.653 0.427 1.0010.0503 78 0.734 0.493 1.092 0.1272 0.706 0.469 1.062 0.0946 76 0.6610.443 0.986 0.0425 0.643 0.427 0.969 0.0347

Association Between High Levels of Anti-OxCL and Risk for Cvd(MI-Stroke) Men

aOxCL Crude Adjust percentile P- P- unit RR 95% CI values RR 95% CIvalues 98 0.750 0.159 3.532 0.7161 0.522 0.104 2.625 0.4301 96 0.9180.336 2.507 0.8672 0.705 0.245 2.024 0.5155 94 0.648 0.264 1.592 0.34380.539 0.211 1.375 0.1959 90 0.415 0.182 0.949 0.0371 0.364 0.157 0.8430.0184 86 0.425 0.211 0.858 0.0170 0.359 0.174 0.743 0.0057 84 0.4210.215 0.821 0.0112 0.358 0.179 0.712 0.0035 82 0.473 0.258 0.868 0.01560.421 0.225 0.787 0.0067 80 0.578 0.334 1.001 0.0505 0.518 0.293 0.9140.0232 78 0.607 0.361 1.020 0.0594 0.565 0.330 0.968 0.0377 76 0.5340.315 0.905 0.0197 0.496 0.286 0.858 0.0121

Association Between High Levels of Anti-OxCL and Risk for Cvd(Infakt-Stroke) Women

aOxCL Crude Adjust percentile P- P- unit RR 95% CI values RR 95% CIvalues 98 0.600 0.070 5.136 0.6410 0.612 0.067 5.572 0.6628 96 0.6520.136 3.133 0.5930 0.696 0.136 3.570 0.6641 94 0.653 0.178 2.391 0.51940.746 0.195 2.851 0.6688 90 1.121 0.475 2.643 0.7944 1.170 0.483 2.8360.7280 86 0.756 0.343 1.663 0.4861 0.716 0.317 1.619 0.4222 84 0.8780.430 1.793 0.7215 0.905 0.435 1.881 0.7889 82 0.865 0.439 1.702 0.67370.878 0.439 1.757 0.7136 80 0.924 0.486 1.755 0.8083 0.893 0.462 1.7270.7371 78 0.992 0.530 1.856 0.9788 0.967 0.510 1.833 0.9179 76 0.9190.491 1.719 0.7921 0.911 0.484 1.717 0.7739

EXAMPLE10

Patient sera from a women control population was used herein. Data wasobtained by use of simple regression and Spearman Rank test was used instatistical analysis (Stat View).

The association between important parameters and determinants inrelation to anti-oxCL is determined.

Controls from the General Population (Population Controls; 26 Women)

Clinical role of antibodies against oxidized cardiolipin and R p Glucosin plasma −0.41 0.035 Systolic blood pressure −0.66 0.001 Diastolicblood pressure −0.428 0.015 Age no association 0.2 BMI −0.56 0.0053

No association with anticardiolipin antibodies or lupus antikoagulans(an indirect measure for anti-cardiolipin antibodies): p=0.41

Thus, these data indicate strong and significant negative associationsbetween anti-OxCL antibodies and important factors as blood pressure,diabetes and blood sugar, Body Mass Index and a positive associationwith endothelial function. One mechanism can be oxCL-effects onendothelium, causing endothelial activation and dysfunction ifprotective anti-oxCL are low.

The role of anti-oxCL in 52 women with a prototypic autoimmune disorder,systemic lupus erythematosus (SLE) is demonstrated.

SLE-Patients: Association Between Anti-OxCL in Women with SLE (n=52)

Clinical role of antibodies against oxidized cardiolipin and R PTNF-induction when leukocytes (PBMC) are cultured −0.32 0.02. withendotoxin 1 ng/ml and serum from patients Endothelial function(nitroinduced dilatation of a 0.43 0.023 Brachialis) VCAM (importantinflammatory/vascular marker): −0.40 0.0046 TNF (Major inflammatorycytokine) −0.40 0.0043 Age no association 0.89 Disease severity indexSLICC: below index 4 (117 ± 48 compared to above index 4 (63 ± 14); p =0.0085

Thus, strong negative association between major SLE measures andinflammation and anti-OxCL exists. In the prototypic autimmune diseaseSLE, a strong negative association between anti-oxCL on the one hand andTNF-induction and other inflammatory markers, is determined, indicatingan anti-inflammatory role played by these antibodies. Further, there isa positive association with endothelial function, indicating a positiveeffect which also has implications for cardiovascular disease. Aspecific potential role in SLE-manifestations and disease severity isdemonstrated by an association with SLICC, an index of disease damage,where higher anti-OxCL could ameliorate disease.

EXAMPLE11

Both CD4 and CD8 positive T-cells from the cell line Human PBMC wherestudied in an incubation setup illustrated in FIG. 11A-F. Both T-celltypes where incubated with, buffer (control), oxCL or CL to determinethe effect on activation, interpreted by the expression of CD69 surfacemolecule after stimulation.

In this experiment the attention must be drawn to the quadrant Q2 of theflow cytometry data shown. This quadrant represents the percentage ofT-cells in the population tested to be CD69 positive, e.g. activated bythe stimuli prior to the data collection.

The control-stimulation (FIG. 11 A and D), without CL or oxCL, noinduction of CD69 expression was detected in CD4 or CD8 positivelymphocytes. The same is true for the population of T-cells treated withCL (FIG. 11 C and F) before measuring the CD69 expression, representedby very similar data plots. However, the expression of CD69 was notablyelevated in oxCL stimulated T-cells positive for CD4 and CD8 (FIG. 11 Band E). A distinct shift from quadrant Q1 to Q2 is seen when comparingdata from the control and CL-experiment to the oxCL-data.

This experiment shows that an immune response can be induced by oxCL,indicated by the oxCL, but not CL mediated expression of CD69 on thesurface of CD4 and CD8 positive T-cells.

FIG. 12A-C illustrates the effect of anti-oxCL on amyloid peptide 1-42induced cell death.

The beta amyloid (Abeta) and especially Abeta peptide (1-42), is animportant component of senile plaques in Alzheimer's disease, and isknown to be directly responsible for the production of free radicalstoxic to brain tissue. Abeta(1-42)-induced free radical oxidative stressin the neurodegeneration observed in AD brain may be one mechanism forneurotoxicity. Human SH-SY5Y neuroblastoma cells were used in cellculture systems.

The cells were treated with 5uM of the peptide for 24 hours. Cell deathwas determined by the addition of 1 mg/mL propidium iodide (PI), whichlabels the nucleus in dying cells which lack an intact plasma membrane.

In the control experiment the amount of dead cells was measured to23,99%, whereas the amlyoid peptide 1-42 induced cell death increasedthat number to 45,54%. When amyloid peptidel-42 was incubated togetherwith oxCL-IgG, 30 ug/ml, (anti-oxCL), the cell dead is reduced to37,10%.

This experiment confirms that anti oxCL will have a protective effect oncell death induced by the amyloid peptide 1-42. Thus, this supports thenotion that anti-OxCL can have a protective effect against Alzheimer.

Thus, from the above it emerges, that oxCL has a T-cell activatingproperties, and is involved in cell death, which is supported by thecell death protective effect of anti-oxCL illustrated in FIG. 12A-C.

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All given references as listed above and discussed in the descriptionare hereby incorporated by reference.

1.-14. (canceled)
 15. A method of treating or reducing the risk ofdeveloping arthritis in a mammal in need thereof, wherein said arthritisis psoriatic arthritis or osteoarthritis, said method comprisingadministering to the mammal a therapeutically effective amount of anagent that inhibits the activity of oxidised cardiolipin (oxCL).
 16. Themethod according to claim 15, wherein the arthritis is psoriaticarthritis.
 17. The method according to claim 15, wherein the arthritisis osteoarthritis.
 18. The method according to claim 15, wherein themammal is selected from the group consisting of mice, rats, rabbits,dogs, cats, cattle, horses and humans.
 19. The method according to claim15, wherein the mammal is a horse.
 20. The method according to claim 15,wherein the mammal is a dog.
 21. The method according to claim 15,wherein the mammal is a human.
 22. The method according to claim 15,wherein the agent inhibits the pro-inflammatory activity of oxCL. 23.The method according to claim 15, wherein the agent that inhibits theactivity of oxCL is selected from the group consisting of Annexin A5, ora monoclonal or polyclonal antibody of isotype IgA, IgD, IgE, IgG, IgM,raised against oxCL or bioactive components and/or parts/fragmentsthereof
 24. The method according to claim 23, wherein the Annexin A5inhibits the pro-inflammatory activity of oxCL.
 25. The method accordingto claim 23, wherein the Annexin A5 binds to phosphatidylserine.
 26. Themethod according to claim 23, wherein the Annexin A5 binds to oxCL. 27.The method according to claim 15, wherein the agent is in combinationwith any suitable adjuvants.